Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis.

BACKGROUND: Necrotic enteritis (NE) is a major enteric disease in poultry, yet effective mitigation strategies remain elusive. Deoxycholic acid (DCA) and butyrate, two major metabolites derived from the intestinal microbiota, have independently been shown to induce host defense peptide (HDP) synthesis. However, the potential synergy between these two compounds remains unexplored. METHODS: To investigate the possible synergistic effect between DCA and butyrate in regulating HDP synthesis and barrier function, we treated chicken HD11 macrophage cells and jejunal explants with DCA and sodium butyrate (NaB), either individually or in combination, for 24 h. Subsequently, we performed RNA isolation and reverse transcription-quantitative PCR to analyze HDP genes as well as the major genes associated with barrier function. To further determine the synergy between DCA and NaB in enhancing NE resistance, we conducted two independent trials with Cobb broiler chicks. In each trial, the diet was supplemented with DCA or NaB on the day-of-hatch, followed by NE induction through sequential challenges with Eimeria maxima and Clostridium perfringens on d 10 and 14, respectively. We recorded animal mortality after infection and assessed intestinal lesions on d 17. The impact of DCA and NaB on the microbiota in the ileum and cecum was evaluated through bacterial 16S rRNA gene sequencing. RESULTS: We found that the combination of DCA and NaB synergistically induced multiple HDP genes in both chicken HD11 cells and jejunal explants. Additionally, the gene for claudin-1, a major tight junction protein, also exhibited synergistic induction in response to DCA and NaB. Furthermore, dietary supplementation with a combination of 0.75 g/kg DCA and 1 g/kg NaB led to a significant improvement in animal survival and a reduction in intestinal lesions compared to either compound alone in a chicken model of NE. Notably, the cecal microbiota of NE-infected chickens showed a marked decrease in SCFA-producing bacteria such as Bacteroides, Faecalibacterium, and Cuneatibacter, with lactobacilli becoming the most dominant species. However, supplementation with DCA and NaB largely restored the intestinal microbiota to healthy levels. CONCLUSIONS: DCA synergizes with NaB to induce HDP and claudin-1 expression and enhance NE resistance, with potential for further development as cost-effective antibiotic alternatives.

Strain specificity of lactobacilli with promoted colonization by galactooligosaccharides administration in protecting intestinal barriers during Salmonella infection.

INTRODUCTION: Galactooligosaccharides (GOS) are lactogenic prebiotics that exert health benefits by stimulating the growth of different Lactobacillus strains in the gastrointestinal (GI) tract. OBJECTIVES: This study aimed to investigate the mechanism of action of different GOS-enriched lactobacilli in intestinal health. METHODS: Piglets and mice were supplemented with GOS to identify specific enrichment of Lactobacillus. The protective effects of individual GOS-enriched lactobacilli were investigated in Salmonella-infected mice. Macrophage depletion and transcriptome analysis were further performed to assess the involvement of macrophages and the underlying mechanisms of individual lactobacilli. An in vitro cell co-culture system was also used to evaluate the anti-adhesive and anti-invasive activities of lactobacilli against Salmonella in epithelial cells. RESULTS: GOS markedly increased the relative abundance of three lactobacilli including L. delbrueckii, L. johnsonii, and L. reuteri in both piglets and mice. Supplementation with GOS further alleviated Salmonella infection in mice. L. delbrueckii (ATCC®BAA 365™), but not L. johnsonii or L. reuteri, enhanced propionate production in the intestinal tract and ameliorated Salmonella-induced intestinal inflammation and barrier dysfunction by suppressing the JAK2-STAT3 signaling and M1 macrophage polarization. L. johnsonii (BNCC 186110), on the other hand, inhibited Salmonella adhesion and invasion of epithelial cells through competitive exclusion. However, L. reuteri (BNCC 186135) failed to protect mice against Salmonella infection. CONCLUSION: GOS-enriched lactobacilli show a differential role in protecting against Salmonella-induced intestinal barrier dysfunction and inflammation. Our results provide novel insights into the mechanism of action of GOS and individual Lactobacillus strains in the control and prevention of intestinal inflammatory disorders.

Mercury Pollution History in Tropical and Subtropical American Lakes: Multiple Impacts and the Possible Relationship with Climate Change.

Sediment cores obtained from 11 tropical and subtropical American lakes revealed that local human activities significantly increased mercury (Hg) inputs and pollution levels. Remote lakes also have been contaminated by anthropogenic Hg through atmospheric depositions. Long-term sediment-core profiles revealed an approximately 3-fold increase in Hg fluxes to sediments from c. 1850 to 2000. Generalized additive models indicate that c. 3-fold increases in Hg fluxes also occurred since 2000 in the remote sites, while Hg emissions from anthropogenic sources have remained relatively stable. The tropical and subtropical Americas are vulnerable to extreme weather events. Air temperatures in this region have shown a marked increase since the 1990s, and extreme weather events arising from climate change have increased. When comparing Hg fluxes to recent (1950-2016) climatic changes, results show marked increases in Hg fluxes to sediments during dry periods. The Standardized Precipitation-Evapotranspiration Index (SPEI) time series indicate a tendency toward more extreme drier conditions across the study region since the mid-1990s, suggesting that instabilities in catchment surfaces caused by climate change are responsible for the elevated Hg flux rates. Drier conditions since c. 2000 appear to be promoting Hg fluxes from catchments to lakes, a process that will likely be exacerbated under future climate-change scenarios.

Epigenetic Regulation of Host Defense Peptide Synthesis: Synergy Between Histone Deacetylase Inhibitors and DNA/Histone Methyltransferase Inhibitors.

Host defense peptides (HDPs) are an integral part of the innate immune system acting as the first line of defense. Modulation of HDP synthesis has emerged as a promising host-directed approach to fight against infections. Inhibition of histone deacetylation or DNA methylation is known to enhance HDP gene expression. In this study, we explored a possible synergy in HDP gene induction between histone deacetylase inhibitors (HDACi) and DNA/histone methyltransferase inhibitors (DNMTi/HMTi). Two chicken macrophage cell lines were treated with structurally distinct HDACi, HMTi, or DNMTi individually or in combinations, followed by HDP gene expression analysis. Each epigenetic compound was found to be capable of inducing HDP expression. To our surprise, a combination of HDACi and HMTi or HDACi and DNMTi showed a strong synergy to induce the expressions of most HDP genes. The HDP-inducing synergy between butyrate, an HDACi, and BIX01294, an HMTi, were further verified in chicken peripheral blood mononuclear cells. Furthermore, tight junction proteins such as claudin 1 were also synergistically induced by HDACi and HMTi. Overall, we conclude that HDP genes are regulated by epigenetic modifications. Strategies to increase histone acetylation while reducing DNA or histone methylation exert a synergistic effect on HDP induction and, therefore, have potential for the control and prevention of infectious diseases.

Biogeography, succession, and origin of the chicken intestinal mycobiome.

BACKGROUND: Extensive work has been accomplished to characterize the intestinal bacterial community, known as the microbiota, and its association with host health and disease. However, very little is known about the spatiotemporal development and the origin of a minor intestinal fungal community, known as the mycobiota, in humans and animals, particularly in avian species. RESULTS: In this study, we comprehensively characterized the biogeography and succession of the gastrointestinal (GI) mycobiota of broiler chickens and further revealed the fungal sources that are responsible for initial and long-term establishment of the mycobiota in the GI tract. Using Illumina sequencing of the internal transcribed spacer 2 (ITS2) region of fungal rRNA genes, we detected significant spatial and temporal differences in the mycobiota along the GI tract. In contrary to the microbiota, the mycobiota was more diverse in the upper than the lower GI tract with no apparent trend of succession up to 42 days of age. The intestinal mycobiota was dominated by the phyla Ascomycota and Basidiomycota with Gibberella, Aspergillus, and Candida being the most abundant genera. Although the chicken mycobiota was highly dynamic, Fusarium pseudonygamai was dominant throughout the GI tract regardless of age in this study. The core chicken mycobiome consisted of 26 fungal taxa accounting for greater than 85% of the fungal population in each GI location. However, we observed high variations of the intestinal mycobiota among different studies. We also showed that the total fungal population varied greatly from 1.0 × 104 to 1.1 × 106 /g digesta along the GI tract and only accounted for less than 0.06% of the bacteria in day-42 broilers. Finally, we revealed that the mycobiota from the hatchery environment was responsible for initial colonization in the GI tract of newly hatched chickens, but was quickly replaced by the fungi in the diet within 3 days. CONCLUSIONS: Relative to the intestinal microbiota that consists of trillions of bacteria in hundreds of different species and becomes relatively stabilized as animals age, the chicken intestinal mycobiota is a minor microbial community that is temporally dynamic with limited diversity and no obvious pattern of successive changes. However, similar to the microbiota, the chicken mycobiota is spatially different along the GI tract, although it is more diverse in the upper than the lower GI tract. Dietary fungi are the major source of the intestinal mycobiota in growing chickens. Video abstract.

Perturbations of the ileal mycobiota by necrotic enteritis in broiler chickens.

BACKGROUND: Intestinal microbiota is critical for maintaining animal health and homeostasis. However, involvement of the fungal community, also known as the mycobiota, in animal health and disease is poorly understood. This study was aimed to examine the association between the intestinal mycobiota and the severity of necrotic enteritis (NE), an economically significant poultry disease. METHODS: A total of 90 day-of-hatch Cobb broilers were infected with Eimeria maxima on d 10, followed by an oral challenge with C. perfringens on d 14 to induce NE, while another 10 broilers were served as mock-infected controls. On d 17, the lesions in the jejunum were scored, and the ileal digesta were subjected to DNA isolation and real-time PCR quantification of total bacterial and fungi populations. Internal transcribed spacer 2 (ITS2) amplicon sequencing was also performed to profile the ileal mycobiota composition. Changes in the ileal mycobiota in response to NE were investigated. Spearman correlation analysis was further conducted to identify the correlations between relative abundances of individual ileal fungi and the severity of NE. RESULTS: While the total bacterial population in the ileum was increased by 2- to 3-fold in NE chickens, the total fungal population was progressively declined in more exacerbated NE, with the most severely infected chickens showing a nearly 50-fold reduction relative to mock-infected controls. Richness of the ileal mycobiota also tended to reduce in chickens with NE (P = 0.06). Compositionally, among 30 most abundant fungal amplicon sequence variants (ASVs), 11 were diminished and 7 were enriched (P < 0.05), while 12 remained largely unchanged in NE-afflicted chickens (P > 0.05). Multiple Wallemia and Aspergillus species were markedly diminished in NE (P < 0.05) and also showed a significant negative correlation with NE severity (P < 0.05). CONCLUSIONS: Dysbiosis of the ileal mycobiota is induced evidently by NE and the extent of the dysbiosis is positively correlated with disease severity. These findings suggest a possible role of the intestinal mycobiota in NE pathogenesis and highlight the mycobiota as a new potential target for NE mitigation in poultry.

Butyrate, Forskolin, and Lactose Synergistically Enhance Disease Resistance by Inducing the Expression of the Genes Involved in Innate Host Defense and Barrier Function.

The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are three natural products known to induce the synthesis of host defense peptides (HDP), which are a critical component of innate immunity. In this study, the synergy among butyrate, forskolin, and lactose in enhancing innate host defense, barrier function, and resistance to necrotic enteritis and coccidiosis was investigated. Our results indicated that the three compounds synergistically augmented the expressions of multiple HDP and barrier function genes in chicken HD11 macrophages. The compounds also showed an obvious synergy in promoting HDP gene expressions in chicken jejunal explants. Dietary supplementation of a combination of 1 g/kg sodium butyrate, 10 mg/kg forskolin-containing plant extract, and 10 g/kg lactose dramatically improved the survival of chickens from 39% to 94% (p < 0.001) in a co-infection model of necrotic enteritis. Furthermore, the three compounds largely reversed growth suppression, significantly alleviated intestinal lesions, and reduced colonization of Clostridium perfringens or Eimeria maxima in chickens with necrotic enteritis and coccidiosis (p < 0.01). Collectively, dietary supplementation of butyrate, forskolin, and lactose is a promising antibiotic alternative approach to disease control and prevention for poultry and possibly other livestock species.

Identification of an Intestinal Microbiota Signature Associated With the Severity of Necrotic Enteritis.

Necrotic enteritis (NE), an economically devastating disease of poultry caused by pathogenic Clostridium perfringens, is known to induce small intestinal lesions and dysbiosis. However, the intestinal microbes that are associated with NE severity are yet to be characterized. Here, we investigated the link between the ileal microbiota and disease severity in a chicken model of clinical NE using 16S rRNA gene sequencing. Our results indicated that richness and Shannon Index of the ileal microbiota were drastically reduced (p<0.01) as NE was exacerbated. While the relative abundance of C. perfringens increased from 0.02% in healthy chickens to 58-70% in chickens with severe infection, a majority of the ileal microbes were markedly diminished, albeit varying in their sensitivity to NE. Compositionally, a large group of ileal microbes showed a significant correlation with NE severity. Firmicutes, such as group A and B Lactobacillus, Lactobacillus reuteri, Subdoligranulum variabile, Mediterraneibacter, and Staphylococcus as well as two genera of Actinobacteria (Corynebacterium and Kocuria) and two highly related Cyanobacteria were progressively declined as NE was aggravated. Other Firmicutes, such as Weissella, Romboutsia, Kurthia, Cuneatibacter, Blautia, and Aerococcus, appeared much more sensitive and were rapidly abolished in chickens even with mild NE. On the other hand, Enterococcus cecorum and two Escherichia/Shigella species were only enriched in the ileal microbiota of chickens with extremely severe NE, while several other species such as Streptococcus gallolyticus and Bacteroides fragilis remained unaltered by NE. Functionally, secondary bile acid biosynthesis was predicted to be suppressed by NE, while biosynthesis of aromatic and branched-amino acids and metabolism of a majority of amino acids were predicted to be enhanced in the ileum of NE-afflicted chickens. These intestinal microbes showing a strong correlation with NE severity may provide important leads for the development of novel diagnostic or therapeutic approaches to NE and possibly other enteric diseases.

Linkage between the intestinal microbiota and residual feed intake in broiler chickens.

BACKGROUND: Intestinal microbiota plays a key role in nutrient digestion and utilization with a profound impact on feed efficiency of livestock animals. However, the intestinal microbes that are critically involved in feed efficiency remain elusive. METHODS: To identify intestinal bacteria associated with residual feed intake (RFI) in chickens, male Cobb broiler chicks were individually housed from day 14 to day 35. Individual RFI values were calculated for 56 chickens. Luminal contents were collected from the ileum, cecum, and cloaca of each animal on day 35. Bacterial DNA was isolated and subjected to 16S rRNA gene sequencing. Intestinal microbiota was classified to the feature level using Deblur and QIIME 2. High and low RFI groups were formed by selecting 15 and 17 chickens with the most extreme RFI values for subsequent LEfSe comparison of the difference in the microbiota. Spearman correlation analysis was further performed to identify correlations between the intestinal microbiota composition and RFI. RESULTS: No significant difference in evenness, richness, and overall diversity of the microbiota in the ileum, cecum, or cloaca was observed between high and low RFI chickens. However, LEfSe analysis revealed a number of bacterial features being differentially enriched in either high or low RFI chickens. Spearman correlation analysis further identified many differentially enriched bacterial features to be significantly correlated with RFI (P < 0.05). Importantly, not all short-chain fatty acid (SCFA) producers showed a positive association with RFI. While two novel members of Oscillibacter and Butyricicoccus were more abundant in low-RFI, high-efficiency chickens, several other SCFA producers such as Subdoligranulum variabile and two related Peptostreptococcaceae members were negatively associated with feed efficiency. Moreover, a few closely-related Lachnospiraceae family members showed a positive correlation with feed efficiency, while others of the same family displayed an opposite relationship. CONCLUSIONS: Our results highlight the complexity of the intestinal microbiota and a need to differentiate the bacteria to the species, subspecies, and even strain levels in order to reveal their true association with feed efficiency. Identification of RFI-associated bacteria provides important leads to manipulate the intestinal microbiota for improving production efficiency, profitability, and sustainability of poultry production.